Two main research directions in our group are synthetic biology and molecular immunology. Synthetic biology combines the engineering approach with biological systems, aiming to introduce new properties into biological or biomimetic systems with potential applications and aiming to understand the function of complex biological systems by constructing them from the bottom up. While the two directions developed independently we find that their combination along with a diversity of experimental techniques is quite powerfull. The main research topics within synthetic biology include
i) designed polypeptide nanostructures,
ii) engineering of mammalian cell signaling and information processing and
iii) gene and cell therapy technologies.
Research on protein nanostructures, funded by an ERC Advanced Grant MaCChines (2018-2024), is focused on coiled-coil-based protein assembly design, named coiled-coil protein origami (CCPO), which is our original platform to design modular proteins based on concatenated coiled-coil dimer-forming segments that self-assemble into a designed shape based on the mathematically defined path. Since the invention of this type of modular protein design, which is distinct from natural globular protein folds, we have advanced this technology by the additional tunable CC modules (Drobnak et al. JACS 2017), new CCPO folds (different tetrahedral topologies, rectangular pyramid and trigonal prism) that self-assemble in mammalian cells and in vivo (in mouse) without triggering any adverse physiological effects (Ljubetič et al., Nat.Biotech. 2017), multi-chain hierarchical assembly of a trigonal bipyramid and its regulation by proteolysis (Lapenta et al., Nat.Comm. 2021), design of the CCPO folding pathways that enables multiple use of the same module within the same chain (Aupič et al., Nat.Comm. 2021), metal ion-regulated assembly of designed modular protein cages (Aupič et al., Sci. Adv. 2022), and preparation, characterization, crystal structure determination of nanobodies binding to designed CC modules and their binding to diverse CCPO polyhedra (Majerle et al., PNAS 2021). Finally, we showed crystal structure of de novo designed CC protein origami triangle (Satler et al., JACS 2023) and applied designed CC-dimer-forming modules to formulate CC-liquid-liquid phase separated condensates in mammalian cells (Ramšak et al., Nat. Comm. 2023).
Coiled-coil modules have also been successfully implemented for the regulation of biological processes in mammalian cells, by introduction of fast signaling pathways based on the proteolysis and coiled-coils, which enables construction of Boolean logic gates in mammalian cells that respond to the chemical signal within few minutes, rather than hours as needed for transcriptional regulation (Fink et al., Nat.Chem.Biol. 2019), multiplexing cellular localization, potent transcriptional regulators and strong amplification of light- and chemical-regulation of transcription by coiled-coils in mammalian cells and concatenated coiled-coil segments (CCCtag) that enable tuning of the response and recruitment of different number of the desired protein domains (Lebar et al., Nat.Chem. Biol. 2020), facilitated polarized displacement of TALEs proteins from DNA, where we demonstrated displacement of a DNA-bound TALE protein by another TALE only in case when it binds to 5’ and demonstrated a new mechanism of fast transcriptional repression (Lebar et al., Nat. Chem. Biol. 2019), regulation of fast secretion or membrane translocation of proteins in mammalian cells by regulated cleavage of the endoplasmic reticulum retention signal and its application for fast secretion of insulin, anti-inflammatory cytokine and regulation of activation of CAR T cells (Praznik et al., Nat. Comm. 2022), recruitment based on the CC-heterodimer of an exonuclease to CRISPR/Cas for enhanced gene editing (Lainšček et al., Nat. Comm. 2022), cell regulation by segmentation strategy of de novo designed four-helical bundles (Merljak et al., Nat. Comm. 2023), and by regulation of proteins, based on inserting a peptide into a loop of a target protein that retains its function (Plaper et al., Cell Discov. 2024).
We approached the regulation of biological processes in mammalian cells also by chemically inducible systems, by engineering inducible split protein regulators in which ligand-binding proteins of human origin are split into two fragments that reassemble in the presence of a physiological ligand or clinically approved drug (Rihtar et al., Nat. Chem. Biol. 2023), and to regulate CD19 CAR-T cell activation based on an engineered downstream NFAT transcription factors, whose activity can be regulated via chemically induced heterodimerization systems (Lainšček et al., Mol. Ther. Oncolytics 2023).
In molecular immunology, we have been mainly focusing on innate immunity, including the mechanism of activation of NLRP3 inflammasome and the role of its LRR domain and showed that activation does not proceed via a nucleation event (Hafner-Bratkovič et al., Nat. Comm. 2018), and the mechanism of inflammatory cell death (pyroptosis) via gasdermin D oligomerization promoted by Ragulator (Evavold et al., Cell 2021). We demonstrated that microbial components and environmental toxins induce oxidative stress that promotes GSDMD pore-forming activity through direct modification of C192 (Devant et al., Cell Reports, 2023). We also demonstrated that gasdermin D makes pores not only in the plasma membrane but also permeabilizes mitochondrial membranes to enhance pyroptosis (Miao et al., Immunity 2023). We investigated the role of oxidative stress in activation of TLR4 via partial oxidation of phospholipids and synergistic activity between phospholipase A2 and 15-lipoxygenase that generates endogenous TLR4/MD2 agonist from extracellular vesicles (Ha et al., PNAS 2020), mechanism of transfer of the signaling complex comprising mutate MyD88 in diffuse B-cell lymphoma, which is frequent in Waldenstrom’s macroglobulinemia (WM) and demonstrates intercellular transfer of proinflammatory signal that is able to engage the endogenous signaling pathway in target cells, which we demonstrated in a bone marrow of a muse model of WM (Manček-Keber et al., Blood 2018). We also investigated the mechanism of SARS-CoV-2 infection and demonstrated that thiol-reducing peptides based on the active site of oxidoreductase thioredoxin 1, called thioredoxin mimetic (TXM) peptides, can prevent syncytia formation, SARS-CoV-2 entry into cells, and subsequent infection (Govednik et al., Antiviral Res. 2023).